Borrelia burgdorferi Engages Mammalian Type I IFN Responses via the cGAS-STING Pathway.

Borrelia burgdorferi, the etiologic agent of Lyme disease, is a spirochete that modulates numerous host pathways to cause a chronic, multisystem inflammatory disease in humans. B. burgdorferi infection can lead to Lyme carditis, neurologic complications, and arthritis because of the ability of specific borrelial strains to disseminate, invade, and drive inflammation. B. burgdorferi elicits type I IFN (IFN-I) responses in mammalian cells and tissues that are associated with the development of severe arthritis or other Lyme-related complications. However, the innate immune sensors and signaling pathways controlling IFN-I induction remain unclear. In this study, we examined whether intracellular nucleic acid sensing is required for the induction of IFN-I to B. burgdorferi. Using fluorescence microscopy, we show that B. burgdorferi associates with mouse and human cells in culture, and we document that internalized spirochetes colocalize with the pattern recognition receptor cyclic GMP-AMP synthase (cGAS). Moreover, we report that IFN-I responses in mouse macrophages and murine embryonic fibroblasts are significantly attenuated in the absence of cGAS or its adaptor stimulator of IFN genes (STING), which function to sense and respond to intracellular DNA. Longitudinal in vivo tracking of bioluminescent B. burgdorferi revealed similar dissemination kinetics and borrelial load in C57BL/6J wild-type, cGAS-deficient, or STING-deficient mice. However, infection-associated tibiotarsal joint pathology and inflammation were modestly reduced in cGAS-deficient compared with wild-type mice. Collectively, these results indicate that the cGAS-STING pathway is a critical mediator of mammalian IFN-I signaling and innate immune responses to B. burgdorferi.

The Borrelia burgdorferi Adenylate Cyclase, CyaB, Is Important for Virulence Factor Production and Mammalian Infection.

Borrelia burgdorferi, the causative agent of Lyme disease, traverses through vastly distinct environments between the tick vector and the multiple phases of the mammalian infection that requires genetic adaptation for the progression of pathogenesis. Borrelial gene expression is highly responsive to changes in specific environmental signals that initiate the RpoS regulon for mammalian adaptation, but the mechanism(s) for direct detection of environmental cues has yet to be identified. Secondary messenger cyclic adenosine monophosphate (cAMP) produced by adenylate cyclase is responsive to environmental signals, such as carbon source and pH, in many bacterial pathogens to promote virulence by altering gene regulation. B. burgdorferi encodes a single non-toxin class IV adenylate cyclase (bb0723, cyaB). This study investigates cyaB expression along with its influence on borrelial virulence regulation and mammalian infectivity. Expression of cyaB was specifically induced with co-incubation of mammalian host cells that was not observed with cultivated tick cells suggesting that cyaB expression is influenced by cellular factor(s) unique to mammalian cell lines. The 3' end of cyaB also encodes a small RNA, SR0623, in the same orientation that overlaps with bb0722. The differential processing of cyaB and SR0623 transcripts may alter the ability to influence function in the form of virulence determinant regulation and infectivity. Two independent cyaB deletion B31 strains were generated in 5A4-NP1 and ML23 backgrounds and complemented with the cyaB ORF alone that truncates SR0623, cyaB with intact SR0623, or cyaB with a mutagenized full-length SR0623 to evaluate the influence on transcriptional and posttranscriptional regulation of borrelial virulence factors and infectivity. In the absence of cyaB, the expression and production of ospC was significantly reduced, while the protein levels for BosR and DbpA were substantially lower than parental strains. Infectivity studies with both independent cyaB mutants demonstrated an attenuated phenotype with reduced colonization of tissues during early disseminated infection. This work suggests that B. burgdorferi utilizes cyaB and potentially cAMP as a regulatory pathway to modulate borrelial gene expression and protein production to promote borrelial virulence and dissemination in the mammalian host.

Live Imaging.

Being able to vizualize a pathogen at a site of interaction with a host is an aesthetically appealing idea and the resulting images can be both informative as well as enjoyable to view. Moreover, the approaches used to derive these images can be powerful in terms of offering data unobtainable by other methods. In this article, we review three primary modalities for live imaging Borrelia spirochetes: whole animal imaging, intravital microscopy and live cell imaging. Each method has strengths and weaknesses, which we review, as well as specific purposes for which they are optimally utilized. Live imaging borriliae is a relatively recent development and there was a need of a review to cover the area. Here, in addition to the methods themselves, we also review areas of spirochete biology that have been significantly impacted by live imaging and present a collection of images associated with the forward motion in the field driven by imaging studies.

Borrelia burgdorferi spatiotemporal regulation of transcriptional regulator bosR and decorin binding protein during murine infection.

Lyme disease, caused by Borrelia burgdorferi, is an inflammatory multistage infection, consisting of localized, disseminated, and persistent disease stages, impacting several organ systems through poorly defined gene regulation mechanisms. The purpose of this study is to further characterize the spatiotemporal transcriptional regulation of B. burgdorferi during mammalian infection of borrelial oxidative stress regulator (bosR) and decorin binding protein (dbpBA) by utilizing bioluminescent B. burgdorferi reporter strains and in vivo imaging. Fluctuating borrelial load was also monitored and used for normalization to evaluate expression levels. bosR transcription is driven by two promoters, Pbb0648 and PbosR, and we focused on the native promoter. bosR expression is low relative to the robustly expressed dbpBA throughout infection. In distal tissues, bosR was the highest in the heart during in the first week whereas dbpBA was readily detectable at all time points with each tissue displaying a distinct expression pattern. This data suggests bosR may have a role in heart colonization and the induction of dbpBA indicates a RpoS independent transcriptional regulation occurring in the mammalian cycle of pathogenesis. These finding demonstrate that B. burgdorferi engages unknown genetic mechanisms to uniquely respond to mammalian tissue environments and/or changing host response over time.

The intergenic small non-coding RNA ittA is required for optimal infectivity and tissue tropism in Borrelia burgdorferi.

Post-transcriptional regulation via small regulatory RNAs (sRNAs) has been implicated in diverse regulatory processes in bacteria, including virulence. One class of sRNAs, termed trans-acting sRNAs, can affect the stability and/or the translational efficiency of regulated transcripts. In this study, we utilized a collaborative approach that employed data from infection with the Borrelia burgdorferi Tn library, coupled with Tn-seq, together with borrelial sRNA and total RNA transcriptomes, to identify an intergenic trans-acting sRNA, which we designate here as ittA for infectivity-associated and tissue-tropic sRNA locus A. The genetic inactivation of ittA resulted in a significant attenuation in infectivity, with decreased spirochetal load in ear, heart, skin and joint tissues. In addition, the ittA mutant did not disseminate to peripheral skin sites or heart tissue, suggesting a role for ittA in regulating a tissue-tropic response. RNA-Seq analysis determined that 19 transcripts were differentially expressed in the ittA mutant relative to its genetic parent, including vraA, bba66, ospD and oms28 (bba74). Subsequent proteomic analyses also showed a significant decrease of OspD and Oms28 (BBA74) proteins. To our knowledge this is the first documented intergenic sRNA that alters the infectivity potential of B. burgdorferi.

Genome-wide screen identifies novel genes required for Borrelia burgdorferi survival in its Ixodes tick vector.

Borrelia burgdorferi, the causative agent of Lyme disease in humans, is maintained in a complex biphasic life cycle, which alternates between tick and vertebrate hosts. To successfully survive and complete its enzootic cycle, B. burgdorferi adapts to diverse hosts by regulating genes required for survival in specific environments. Here we describe the first ever use of transposon insertion sequencing (Tn-seq) to identify genes required for B. burgdorferi survival in its tick host. We found that insertions into 46 genes resulted in a complete loss of recovery of mutants from larval Ixodes ticks. Insertions in an additional 56 genes resulted in a >90% decrease in fitness. The screen identified both previously known and new genes important for larval tick survival. Almost half of the genes required for survival in the tick encode proteins of unknown function, while a significant portion (over 20%) encode membrane-associated proteins or lipoproteins. We validated the results of the screen for five Tn mutants by performing individual competition assays using mutant and complemented strains. To better understand the role of one of these genes in tick survival, we conducted mechanistic studies of bb0017, a gene previously shown to be required for resistance against oxidative stress. In this study we show that BB0017 affects the regulation of key borrelial virulence determinants. The application of Tn-seq to in vivo screening of B. burgdorferi in its natural vector is a powerful tool that can be used to address many different aspects of the host pathogen interaction.

Detection of Bioluminescent Borrelia burgdorferi from In Vitro Cultivation and During Murine Infection.

Borrelia burgdorferi, etiologic agent of Lyme disease, is the leading tick-borne disease in the United States with approximately 300,000 cases diagnosed annually. Disease occurs in stages beginning localized infection at the site of a tick bite and progresses to disseminated infection when antibiotic treatment is not administered in a timely manner. A multi-systemic infection develops following dissemination to numerous immunoprotective tissues, such as the heart, bladder, and joints, resulting in late Lyme disease. B. burgdorferi undergoes dynamic genetic regulation throughout mammalian infection and defining the exact role of virulence genes at distinct stages of disease is challenging. The murine model allows for the characterization of the pathogenic function of genes in B. burgdorferi, but traditional end point studies limit the ability to gather data throughout an infection study and greatly increase the required number of mice. Molecular genetic techniques to evaluate and quantitate B. burgdorferi infection are laborious and costly. To partly circumvent these issues, a codon optimized firefly luciferase, under the control of a constitutive borrelial promoter, was introduced into B. burgdorferi enabling the characterization of mutant or modified strains under in vitro growth conditions and throughout murine infection. The detection of bioluminescent B. burgdorferi is highly sensitive and allows for the repeated real-time quantitative evaluation of borrelial load during murine infection. Furthermore, bioluminescence has also been utilized to evaluate alteration in tissue localization and tissue-specific gene expression of B. burgdorferi. In this chapter, we describe the generation of bioluminescent borrelial strains along with methods for in vitro, in vivo, and ex vivo B. burgdorferi studies.

Borrelia burgdorferi Keeps Moving and Carries on: A Review of Borrelial Dissemination and Invasion.

Borrelia burgdorferi is the etiological agent of Lyme disease, a multisystemic, multistage, inflammatory infection resulting in patients experiencing cardiac, neurological, and arthritic complications when not treated with antibiotics shortly after exposure. The spirochetal bacterium transmits through the Ixodes vector colonizing the dermis of a mammalian host prior to hematogenous dissemination and invasion of distal tissues all the while combating the immune response as it traverses through its pathogenic lifecycle. The innate immune response controls the borrelial burden in the dermis, but is unable to clear the infection and thereby prevent progression of disease. Dissemination in the mammalian host requires temporal regulation of virulence determinants to allow for vascular interactions, invasion, and colonization of distal tissues. Virulence determinants and/or adhesins are highly heterogenetic among environmental B. burgdorferi strains with particular genotypes being associated with the ability to disseminate to specific tissues and the severity of disease, but fail to generate cross-protective immunity between borrelial strains. The unique motility of B. burgdorferi rendered by the endoflagella serves a vital function for dissemination and protection from immune recognition. Progress has been made toward understanding the chemotactic regulation coordinating the activity of the two polar localized flagellar motors and their role in borrelial virulence, but this regulation is not yet fully understood. Distinct states of motility allow for dynamic interactions between several B. burgdorferi adhesins and host targets that play roles in transendothelial migration. Transmigration across endothelial and blood-brain barriers allows for the invasion of tissues and elicits localized immune responses. The invasive nature of B. burgdorferi is lacking in proactive mechanisms to modulate disease, such as secretion systems and toxins, but recent work has shown degradation of host extracellular matrices by B. burgdorferi contributes to the invasive capabilities of the pathogen. Additionally, B. burgdorferi may use invasion of eukaryotic cells for immune evasion and protection against environmental stresses. This review provides an overview of B. burgdorferi mechanisms for dissemination and invasion in the mammalian host, which are essential for pathogenesis and the development of persistent infection.

A high-throughput genetic screen identifies previously uncharacterized Borrelia burgdorferi genes important for resistance against reactive oxygen and nitrogen species.

Borrelia burgdorferi, the causative agent of Lyme disease in humans, is exposed to reactive oxygen and nitrogen species (ROS and RNS) in both the tick vector and vertebrate reservoir hosts. B. burgdorferi contains a limited repertoire of canonical oxidative stress response genes, suggesting that novel gene functions may be important for protection of B. burgdorferi against ROS or RNS exposure. Here, we use transposon insertion sequencing (Tn-seq) to conduct an unbiased search for genes involved in resistance to nitric oxide, hydrogen peroxide, and tertiary-butyl hydroperoxide in vitro. The screens identified 66 genes whose disruption resulted in increased susceptibility to at least one of the stressors. These genes include previously characterized mediators of ROS and RNS resistance (including components of the nucleotide excision repair pathway and a subunit of a riboflavin transporter), as well as novel putative resistance candidates. DNA repair mutants were among the most sensitive to RNS in the Tn-seq screen, and survival assays with individual Tn mutants confirmed that the putative ribonuclease BB0839 is involved in resistance to nitric oxide. In contrast, mutants lacking predicted inner membrane proteins or transporters were among the most sensitive to ROS, and the contribution of three such membrane proteins (BB0017, BB0164, and BB0202) to ROS sensitivity was confirmed using individual Tn mutants and complemented strains. Further analysis showed that levels of intracellular manganese are significantly reduced in the Tn::bb0164 mutant, identifying a novel role for BB0164 in B. burgdorferi manganese homeostasis. Infection of C57BL/6 and gp91phox-/- mice with a mini-library of 39 Tn mutants showed that many of the genes identified in the in vitro screens are required for infectivity in mice. Collectively, our data provide insight into how B. burgdorferi responds to ROS and RNS and suggests that this response is relevant to the in vivo success of the organism.

In Vivo Imaging Demonstrates That Borrelia burgdorferi ospC Is Uniquely Expressed Temporally and Spatially throughout Experimental Infection.

Borrelia burgdorferi is a spirochetal bacterium transmitted by the Ixodes tick that causes Lyme disease in humans due to its ability to evade the host immune response and disseminate to multiple immunoprotective tissues. The pathogen undergoes dynamic genetic alterations important for adaptation from the tick vector to the mammalian host, but little is known regarding the changes at the transcriptional level within the distal tissues they colonize. In this study, B. burgdorferi infection and gene expression of the essential virulence determinant ospC was quantitatively monitored in a spatial and temporal manner utilizing reporter bioluminescent borrelial strains with in vivo and ex vivo imaging. Although expressed from a shuttle vector, the PospC-luc construct exhibited a similar expression pattern relative to native ospC. Bacterial burden in skin, inguinal lymph node, heart, bladder and tibiotarsal joint varied between tissues and fluctuated over the course of infection possibly in response to unique cues of each microenvironment. Expression of ospC, when normalized for changes in bacterial load, presented unique profiles in murine tissues at different time points. The inguinal lymph node was infected with a significant B. burgdorferi burden, but showed minimal ospC expression. B. burgdorferi infected skin and heart induced expression of ospC early during infection while the bladder and tibiotarsal joint continued to display PospC driven luminescence throughout the 21 day time course. Localized skin borrelial burden increased dramatically in the first 96 hours following inoculation, which was not paralleled with an increase in ospC expression, despite the requirement of ospC for dermal colonization. Quantitation of bioluminescence representing ospC expression in individual tissues was validated by qRT-PCR of the native ospC transcript. Taken together, the temporal regulation of ospC expression in distal tissues suggests a role for this virulence determinant beyond early infection.

The BBA33 lipoprotein binds collagen and impacts Borrelia burgdorferi pathogenesis.

Borrelia burgdorferi, the etiologic agent of Lyme disease, adapts to the mammalian hosts by differentially expressing several genes in the BosR and Rrp2-RpoN-RpoS dependent pathways, resulting in a distinct protein profile relative to that seen for survival in the Ixodes spp. tick. Previous studies indicate that a putative lipoprotein, BBA33, is produced in an RpoS-dependent manner under conditions that mimic the mammalian component of the borrelial lifecycle. However, the significance and function for BBA33 is not known. Given its linkage to the BosR/Rrp2-RpoN-RpoS regulatory cascade, we hypothesized that BBA33 facilitates B. burgdorferi infection in the mammalian host. The deletion of bba33 eliminated B. burgdorferi infectivity in C3H mice, which was rescued by genetic complementation with intact bba33. With regard to function, a combinatorial peptide approach, coupled with subsequent in vitro binding assays, indicated that BBA33 binds to collagen type VI and, to a lesser extent, collagen type IV. Whole cell binding assays demonstrated BBA33-dependent binding to human collagen type VI. Taken together, these results suggest that BBA33 interacts with collagenous structures and may function as an adhesin in a process that is required to prevent bacterial clearance.

pncA and bptA are not sufficient to complement Ixodes scapularis colonization and persistence by Borrelia burgdorferi in a linear plasmid lp25-deficient background.

The complex segmented genome of Borrelia burgdorferi is comprised of a linear chromosome along with numerous linear and circular plasmids essential for tick and/or mammalian infectivity. The pathogenic necessity for specific borrelial plasmids has been identified; most notably, infections of the tick vector and mammalian host both require linear plasmid 25 (lp25). Genes carried on lp25, specifically bptA and pncA, are postulated to play a role for B. burgdorferi to infect and persist in Ixodes ticks. In this study, we complemented an lp25-deficient borrelial strain with pncA alone or pncA accompanied by bptA to evaluate the ability of the complemented strains to restore larval colonization and persistence through transstadial transmission relative to that of wild-type B. burgdorferi. The acquisition of the complemented strains by tick larvae from infected mice and/or the survival of these strains was significantly decreased when assayed by cultivation and quantitative PCR (qPCR). Only 10% of the pncA-complemented strain organisms were found by culture to survive 17 days following larval feeding, while 45% of the pncA- and bptA-complemented strain organisms survived, with similar results by PCR. However, neither of the complemented B. burgdorferi strains was capable of persisting through the molt to the nymphal stage as analyzed by culture. qPCR analyses of unfed nymphs detected B. burgdorferi genomes in several nymphs at low copy numbers, likely indicating the presence of DNA from dead or dying cells. Overall, the data indicate that pncA and bptA cannot independently support infection, suggesting that lp25 carries additional gene(s) or regulatory elements critical for B. burgdorferi survival and pathogenesis in the Ixodes vector.

The BB0646 protein demonstrates lipase and haemolytic activity associated with Borrelia burgdorferi, the aetiological agent of Lyme disease.

The etiological agent of Lyme disease, Borrelia burgdorferi, is transmitted by ticks of the Ixodes genus and, if untreated, can cause significant morbidity in affected individuals. Recent reports have shown that polyunsaturated fatty acids in the B. burgdorferi cell envelope are potential targets for oxidative damage, which can be lethal. How B. burgdorferi responds to this assault is not known. Herein we report evidence that bb0646 codes for a lipase that is located within the bosR operon and that has specificity for both saturated and polyunsaturated fatty acids. Specifically, strains harbouring mutated copies of the lipase, either in the form of an insertionally inactivated construct or site-directed mutations within the active site, demonstrated attenuated lipolytic and haemolytic phenotypes when compared with the isogenic parent and trans-complements. In vivo analysis showed that while the bb0646 mutant remains infectious, the spirochaetal load is significantly lower than both the isogenic parent and the complemented mutant strains. Taken together, these data demonstrate that BB0646 is a broad substrate specific lipase that contributes to lipolytic and haemolytic activity in vitro and is required for optimal B. burgdorferi infection.

Bioluminescent imaging of Borrelia burgdorferi in vivo demonstrates that the fibronectin-binding protein BBK32 is required for optimal infectivity.

The aetiological agent of Lyme disease, Borrelia burgdorferi, is transmitted via infected Ixodes spp. ticks. Infection, if untreated, results in dissemination to multiple tissues and significant morbidity. Recent developments in bioluminescence technology allow in vivo imaging and quantification of pathogenic organisms during infection. Herein, luciferase-expressing B. burgdorferi and strains lacking the decorin adhesins DbpA and DbpB, as well as the fibronectin adhesin BBK32, were quantified by bioluminescent imaging to further evaluate their pathogenic potential in infected mice. Quantification of bacterial load was verified by quantitative PCR (qPCR) and cultivation. B. burgdorferi lacking DbpA and DbpB were only seen at the 1 h time point post infection, consistent with its low infectivity phenotype. The bbk32 mutant exhibited a significant decrease in its infectious load at day 7 relative to its parent. This effect was most pronounced at lower inocula and imaging correlated well with qPCR data. These data suggest that BBK32-mediated binding plays an important role in B. burgdorferi colonization. As such, in vivo imaging of bioluminescent Borrelia provides a sensitive means to detect, quantify and temporally characterize borrelial dissemination in a non-invasive, physiologically relevant environment and, more importantly, demonstrated a quantifiable infectivity defect for the bbk32 mutant.

Genetic transformation of Borrelia burgdorferi.

The development of robust genetic tools to manipulate Borrelia burgdorferi, the etiologic agent of Lyme disease, now allows investigators to assess the role(s) of individual genes in the context of experimental Lyme borreliosis. This unit is devoted to the description of experimental approaches that are available for the molecular genetic analysis of B. burgdorferi with an emphasis on cultivation, electrotransformation, selection of desired mutants, and genetic complementation of acquired mutants. The intent is to provide a consensus protocol that encapsulates the methodologies currently employed by the B. burgdorferi research community and describe pertinent issues that must be accounted for when working with these pathogenic spirochetal bacteria.

Characterization of a conditional bosR mutant in Borrelia burgdorferi.

Borrelia burgdorferi, the etiological agent of Lyme disease, adapts to unique host environments as a consequence of its complex life cycle that spans both arthropod and mammalian species. In this regard, B. burgdorferi must adapt to various environmental signals, pHs, temperatures, and O(2) and CO(2) levels to establish infectious foci. We hypothesize that the BosR protein functions as a global regulator that is required for both borrelial oxidative homeostasis and pathogenesis. To assess the role of BosR in B. burgdorferi, we constructed an IPTG (isopropyl-beta-d-thiogalactopyranoside)-regulated bosR strain. The selective decrease of bosR resulted in a change in growth when cells were cultured either anaerobically or microaerobically; however, a distinct growth defect was observed for anaerobically grown B. burgdorferi relative to the growth attenuation observed for microaerobically grown B. burgdorferi. B. burgdorferi cells in which BosR levels were reduced were more sensitive to hydrogen peroxide and produced lower levels of NapA (Dps) and SodA, proteins involved in the oxidative stress response. In addition, the levels of OspC and DbpA were also induced coincident with increased BosR levels, suggesting that BosR interfaces with the RpoS regulatory cascade, which is known to modulate virulence gene expression in B. burgdorferi. Taken together, these results indicate that BosR is involved in the resistance of B. burgdorferi to oxidative stressors and affects the expression of genes, either directly or indirectly, whose products are important in borrelial pathogenesis.

The BosR regulatory protein of Borrelia burgdorferi interfaces with the RpoS regulatory pathway and modulates both the oxidative stress response and pathogenic properties of the Lyme disease spirochete.

Summary Borrelia burgdorferi, the Lyme disease spirochete, adapts as it moves between the arthropod and mammalian hosts that it infects. We hypothesize that BosR serves as a global regulator in B. burgdorferi to modulate the oxidative stress response and adapt to mammalian hosts. To test this hypothesis, a bosR mutant in a low-passage B. burgdorferi isolate was constructed. The resulting bosR::kan(R) strain was altered when grown microaerobically or anaerobically suggesting that BosR is required for optimal replication under both growth conditions. The absence of BosR increased the sensitivity of B. burgdorferi to hydrogen peroxide and reduced the synthesis of Cdr and NapA, proteins important for cellular redox balance and the oxidative stress response, respectively, suggesting an important role for BosR in borrelial oxidative homeostasis. For the bosR mutant, the production of RpoS was abrogated and resulted in the loss of OspC and DbpA, suggesting that BosR interfaces with the Rrp2-RpoN-RpoS regulatory cascade. Consistent with the linkage to RpoS, cells lacking bosR were non-infectious in the mouse model of infection. These results indicate that BosR is required for resistance to oxidative stressors and provides a regulatory response that is necessary for B. burgdorferi pathogenesis.

Borrelia burgdorferi alters its gene expression and antigenic profile in response to CO2 levels.

The etiologic agent of Lyme disease, Borrelia burgdorferi, must adapt to the distinct environments of its arthropod vector and mammalian host during its complex life cycle. B. burgdorferi alters gene expression and protein synthesis in response to temperature, pH, and other uncharacterized environmental factors. The hypothesis tested in this study is that dissolved gases, including CO(2), serve as a signal for B. burgdorferi to alter protein production and gene expression. In this study we focused on characterization of in vitro anaerobic (5% CO(2), 3% H(2), 0.087 ppm O(2)) and microaerophilic (1% CO(2), 3.48 ppm O(2)) growth conditions and how they modulate protein synthesis and gene expression in B. burgdorferi. Higher levels of several immunoreactive proteins, including BosR, NapA, DbpA, OspC, BBK32, and RpoS, were synthesized under anaerobic conditions. Previous studies demonstrated that lower levels of NapA were produced when microaerophilic cultures were purged with nitrogen gas to displace oxygen and CO(2). In this study we identified CO(2) as a factor contributing to the observed change in NapA synthesis. Specifically, a reduction in the level of dissolved CO(2), independent of O(2) levels, resulted in reduced NapA synthesis. BosR, DbpA, OspC, and RpoS synthesis was also decreased with the displacement of CO(2). Quantitative reverse transcription-PCR indicated that the levels of the dbpA, ospC, and BBK32 transcripts are increased in the presence of CO(2), indicating that these putative borrelial virulence determinants are regulated at the transcriptional level. Thus, dissolved CO(2) may be an additional cue for borrelial host adaptation and gene regulation.

Transcriptional profiling of Borrelia burgdorferi containing a unique bosR allele identifies a putative oxidative stress regulon.

Borrelia burgdorferi regulates gene expression in response to environmental conditions, including temperature, pH, redox potential and host factors. B. burgdorferi encodes a PerR homologue designated BosR, which presumably serves as a global regulator of genes involved in the oxidative stress response. Infectious B. burgdorferi strain B31 is resistant to oxidative stressors in vitro, whereas the non-infectious isolate was sensitive due, in part, to a point mutation that converts an arginine to a lysine at residue 39 of BosR. Subsequent insertional inactivation of this bosRR39K allele (bosRR39K : : kan(R)) restored resistance to oxidative stressors. These observations suggest that the B. burgdorferi non-infectious bosRR39K : : kan(R) strain may transcribe genes that are also expressed in infectious B. burgdorferi cells, but are repressed in the bosRR39K background, thus explaining the different oxidative stress phenotypes observed between these isolates. To test this hypothesis, macroarray technology and quantitative RT-PCR were utilized to compare the transcriptional profiles from the isogenic bosRR39K and bosRR39K : : kan(R) isolates. Array data indicated that 88 ORFs were significantly expressed in the absence of BosRR39K. Since most affected genes mapped to the chromosome, it is likely that these genes define an important physiologic response for B. burgdorferi. Included within the genes identified was the detoxification gene sodA, as well as other loci not overtly linked to oxidative stress. These results suggest that a putative BosR regulon, as defined by the bosRR39K allele, is required to combat toxic oxidative intermediates, but may also be involved in adaptive strategies that are independent of reactive oxygen species.

A conservative amino acid change alters the function of BosR, the redox regulator of Borrelia burgdorferi.

Borrelia burgdorferi, the aetiologic agent of Lyme disease, modulates gene expression in response to changes imposed by its arthropod vector and mammalian hosts. As reactive oxygen species (ROS) are known to vary in these environments, we asked how B. burgdorferi responds to oxidative stress. The B. burgdorferi genome encodes a PerR homologue (recently designated BosR) that represses the oxidative stress response in other bacteria, suggesting a similar function in B. burgdorferi. When we tested the sensitivity of B. burgdorferi to ROS, one clonal non-infectious B. burgdorferi isolate exhibited hypersensitivity to t-butyl hydroperoxide when compared with infectious B. burgdorferi and other non-infectious isolates. Sequence analysis indicated that the hypersensitive non-infectious isolates bosR allele contained a single nucleotide substitution, converting an arginine to a lysine (bosRR39K). Mutants in bosRR39K exhibited an increase in resistance to oxidative stressors when compared with the parental non-infectious strain, suggesting that BosRR39K functioned as a repressor. Complementation with bosRR39K and bosR resulted in differential sensitivity to t-butyl hydroperoxide, indicating that these alleles are functionally distinct. In contrast to BosR, BosRR39K did not activate transcription of a napA promoter-lacZ reporter in Escherichia coli nor bind the napA promoter/operator domain. However, we found that both BosR and BosRR39K bound to the putative promoter/operator region of superoxide dismutase (sodA). In addition, we determined that cells lacking BosRR39K synthesized fourfold greater levels of the decorin binding adhesin DbpA suggesting that BosRR39K regulates genes unrelated to oxidative stress. Based on these data, we propose that the single amino acid substitution, R39K, dramatically alters the activity of BosR by altering its ability to bind DNA at target regulatory sequences.